This invention relates generally to a rapid one-reagent method for sample preparation for improved electrooptical methods for measuring human and animal erythrocyte volumes and cell hemoglobin contents. More particularly, it relates to a one step method for treating mammalian red blood cells in a sample which can be effectively measured electrooptically for determination of red blood cell volumes whereby the one-step, single reagent treatment provides isovolumetric sphering of erythrocytes in the sample independent of erythrocyte concentration in the original blood sample and without inducing lysis.
Prior art methods utilizing the measured amount of light scattered from individual red cells (erythrocytes) to determine the individual and mean volumes of red cells were subject to error for two reasons: since the native human red cell is a biconcave disc, the amount of light scattered will vary with the orientation of the cell with respect to the incident light beam; and, the shape of the cells can change during dilution and pumping steps.
For a discussion of the above, see Hemolysis and Related Phenomena, Chapter II, pp 10-49 by Eric Ponder (1948) and Transformation and Restoration of Biconcave Shape of Human Erythrocytes Induced by Amphilic Agents and Changes of Ionic Environment, Biochemica et Biophy. Acta, Bernard Deuticke, pp 494-500 (1968).
In Ornstein et al U.S. Pat. No. 4,412,004, commonly assigned, a method is disclosed and claimed which eliminates both of the above-described causes of error and permits a vastly improved method for determination of human red blood cell volumes. In the '004 patent, the inventive method involves a two-step procedure of first sphering erythrocytes isovolumetrically and then fixing isotonically. The method was devised as a two-step procedure in order to be assured that sphering had reached completion before the fixing process began, otherwise, cells might become "stiffened" by cross-linking which occurs during fixation and thus became "frozen" in some intermediate shape between the native biconcave shape and the desired spherical shape. Fixing immediately after sphering permitted the use of higher sphering agent/protein ratios in the fixing solution because cells which might otherwise lyse, were fixed before that could happen. This leads to a more convenient implementation for automated clinical use.
It has now been found, quite unexpectedly, that a single reagent containing appropriate and limited ranges of concentrations of both sphering agent and fixing agent can achieve complete isovolumetric sphering of all erythrocytes of a large range of clinical samples of blood, independently of the concentration of erythrocytes in the whole blood sample, and without inducing lysis of any of the erythrocytes in any of the diluted blood samples. It was not obvious to one of ordinary skill in the art that the employment of particular parameters in a one-step method as disclosed and claimed herein could allow for a procedure in which the speed of the sphering step remains sufficiently greater than the fixing process so that the cells are completely sphered before they have appreciably stiffened. This leads to a faster and still more convenient implementation for automated clinical use.